Whether fleeting or stable, normal or aberrant, protein interactions and their sites of contact form the basis for discovery of biological pathways, disease mechanisms, and opportunities for therapeutic intervention. The goal of this proposal is to intertwine chemistry, biology, and medicine to create a transformative high- throughput technology that precisely identifies protein targets and their explicit sites of interaction. Like the teeth of a key that perfectly fit into a lock, complementary protein shape is critical to the execution of biological interactions. The molecular handshakes of proteins rely on their discrete substructures and these contact points are typically embedded within a complex protein that provides the infrastructure to maintain the essential bioactive fold. Ideally, these evolutionarily honed substructures could be used to capture and thereby catalogue their protein targets; however, out of context from the whole protein, bioactive subdomains often unfold, resulting in loss of biological shape, potency, and specificity. To reclaim the enormous capacity of structured peptides to selectively bind and capture their protein targets, we will first restore their bioactive shape and then chemically derivatize them for both non-covalent and covalent capture. In this proposal, we focus on the peptide 1-helix, arguably the most ubiquitous and versatile biological shape harnessed by the cell. We will apply our robust hydrocarbon stapling technology to synthesize a diversity of bioactive 1-helices and then chemically install new immobilization and intercalating functionalities to expand our grasp of the interactome by trapping the full-range of stable to transient protein interactors. Our covalent capture chemistry and proteomic analyses afford a two-for-one advantage: identification of protein targets and their sites of interaction. Since protein interaction sites are the topographic templates for drug design, we believe that the binding site identification feature of our approach will provide a critical link between interactome discovery and clinical translation. To accomplish our goals, we will take a step-wise approach: (1) structural stabilization, (2) directional affinity capture, (3) covalent capture, and (4) binding site identification. Each step will be adapted for high-throughput and validated using proof-of-concept biological experiments. Once identified and catalogued, protein interactions must be validated biologically. A seminal feature of our approach is that the very 1-helices we use to capture the protein interactome can be used to validate and drug the interactions in cellular and in vivo studies. Thus, we believe that engineering stapled peptides for protein capture will create a powerful and versatile approach to elucidating the interactome, and massively expand the potential for discovery of novel interactions and how they impact health and disease.